The fermentative production of succinic acid (SA) from biomass has already drawn much attention because said acid represents an important constituent of synthetic resins or is a source of further valuable low-molecular chemical compounds, in particular tetrahydrofuran (THF), 1,4-butanediol (BDO), gamma-butyrolactone (GBL) and pyrrolidones (WO-A-2006/066839).
A SA-producing bacterium isolated from bovine rumen was described by Lee et al (2002a). The bacterium is a non-motile, non-spore-forming, mesophilic and capnophilic gram-negative rod or coccobacillus. Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to genus Mannheimia as a novel species, and has been named Mannheimia succiniciproducens MBEL55E. Under 100% CO2 conditions, it grows well in the pH range of 6.0-7.5 and produces SA, acetic acid and formic acid at a constant ratio of 2:1:1. When M. succiniciproducens MBEL55E was cultured anaerobically under CO2— saturation with glucose as carbon source, 19.8 g/L of glucose were consumed and 13.3 g/L of SA were produced in 7.5 h of incubation. Furthermore in this microorganism the production of SA was improved by mutation/deletion of metabolic genes. The combined mutation/deletion of the genes lactate dehydrogenase ldhA, pyruvate-formate-lyase pflB, phosphotransacetylase pta, and acetate kinase ackA genes resulted in a strain converting carbon to SA with a yield (YP/S) of 0.6 g SA per g of carbon source added. The space-time yield for the production of SA was found to be 1.8 g/liter/h. (Lee 2006)
Lin et at 2005 describe a mutant strain of E. coli carrying mutations in the ldh as well as in the pfl genes, described as SB202. However this strain was characterized by slow growth and the inability to ferment a saccharide to completion under anaerobic conditions. Inactive ldh and pfl did cause the carbon flux to bottle up at the pyruvate node, causing pyruvate to accumulate as the major product. In this respect the carbon yield (YP/S) of succinate on the carbon source was found to be lower than 0.15 g/g SA/Carbon.
Sanchez et al. 2005 describe E. coli strains carrying mutations in the ldh, the adhE, ack-pta and iclR genes. In these experiments cells were grown aerobically on complex medium, harvested, concentrated and incubated with carbon sources under anaerobic conditions. Under these specific conditions for the direct conversion of a carbohydrate to SA, carbon yields YP/S of 0.98 to 1.13 g SA per g carbon source were found, with a space-time yield of 0.79 g/l h SA. The carbon utilization for the biomass generation prior to the anaerobic conversion phase has been explicitly not included in this calculation and is not further described.
Hong and Lee (2001) describe E. coli strains carrying mutations in the ldh and pfl genes. These strains do produce SA from the fermentation of carbohydrate, however, with slow carbohydrate utilization and low space-time and carbon yields (YP/S) of SA from the carbohydrate carbon source glucose. In addition succinic, acetic and lactic acid were produced in a ratio of 1:0.034:1.6. In this analysis the growth of the strain carrying mutations in the ldh and pfl genes was retarded if compared to the unmutated parental strain.
Zhu et al. 2005 describe a E. coli strain, mutated in the pfl gene which did not produce succinic acid but lactate and showed poor growth when grown on glucose as the sole substrate.
A significant drawback of the organism Mannheimia sucaniciproducens is, however, its inability to metabolize glycerol, which, as a constituent of triacyl glycerols (TAGs), becomes readily available e. g. as by-product in the transesterification reaction of Biodiesel production (Dharmadi et al., 2006).
The fermentative production of SA from glycerol has been described in the scientific literature (Lee et al., 2001; Dharmadi et al., 2006) and with glycerol higher yields [mass of SA produced/mass of raw material consumed] than with common sugars like glucose were achieved (Lee et al., 2001). However, the space-time yield obtained with glycerol was substantially lower than with glucose (0.14 vs. 1.0 g SA/[L h]).
Only in a few cases anaerobic metabolization of glycerol to fermentation products have been described. E. coli is able to ferment glycerol under very specific conditions such as acidic pH, avoiding accumulation of the fermentation gas hydrogen, and appropriate medium composition (Dharmadi et al 2006, Yazdani and Gonzalez 2007). Many microorganisms are able to metabolize glycerol in the presence of external electron acceptors (respiratory metabolism), few are able to do so fermentatively (i.e. in the absence of electron acceptors). The fermentative metabolism of glycerol has been studied in great detail in several species of the Enterobacteriaceae family, such as Citrobacter freundii and Klebsiella pneumoniae. Dissimilation of glycerol in these organisms is strictly linked to their capacity to synthesize the highly reduced product 1,3-propanediol (1,3-PDO) (Dharmadi et al 2006). The conversion of glycerol into SA using Anaerobiospirillum succiniciproducens has been reported (Lee et al. 2001). This study demonstrated that SA could be produced with little formation of by-product acetic acid by using glycerol as a carbon source, thus facilitating purification of SA. The highest yield was obtained by intermittently feeding glycerol and yeast extract, a strategy that resulted in the production of about 19 g/L of SA. It was noted, however, that unidentified nutritional components present in yeast extract were needed for glycerol fermentation to take place. Saccharides, however, theoretically can be converted to SA with a significantly lower yield than glycerol due to the lower reduction state of saccharides compared to the polyol glycerol. The combination of saccharides with glycerol have been found to function in an SA producing anaerobic organisms (Lee et al. 2001), however without reaching SA titers beyond 29 g/l. In addition the carbon yield YP/S of was found to be only 92% and the SA/AA relation was found to be 4.9:1. Only 4 g/l glycerol were converted to succinic acid at most.
Carboxylation reactions of oxaloacetate catalyzed by the enzymes phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PycA) are utilizing HCO3− as a source of CO2 (Peters-Wendisch, P G et al 1996, 1998). Therefore hydrogencarbonate sources such as NaHCO3, KHCO3, NH4HCO3 and so on can be applied to fermentation and cultivation media to improve the availability of HCO3− in the metabolization of substrates to SA. The production of SA from glucose has not been found to be dependent on the addition of HCO3− in the prior art so far.
Biomass production by anaerobic organisms is limited by the amount of ATP produced from fermentative pathways. Biomass yield of glycerol in anaerobic organisms is lower than of saccharides, like hexoses such as glucose, fructose, pentoses such as xylose arabinose or disaccharides such as sucrose or maltose (Lee et al. 2001, Dharmadi 2007).
Earlier patent application PCT/EP2008/006714, the content of which is herewith incorporated by reference, discloses a bacterial strain, being a member of the family of Pasteurellaceae, originally isolated from rumen, and capable of utilizing glycerol as a carbon source and variant and mutant strains derived there from retaining said capability, in particular, a bacterial strain designated DD1 as deposited with DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) having the deposit number DSM 18541 (ID 06-614) and having the ability to produce succinic acid and variant or mutant strains derived there from retaining at least said ability to produce succinic acid. The DD1 strain belongs to the species Basfia succiniciproducens and the family of Pasteurellaceae as classified by Kuhnert et al., 2010.
There is, therefore, a need for novel bacterial strains, which have the ability to produce organic acids, in particular SA, from glycerol. In particular, such strains should produce said acids with high productivity from glycerol, especially if crude glycerol e. g. from bio diesel production can be used without prior purification. It is an object of the present invention to provide such novel strains and production processes.